Canine sperm vitrification with sucrose: effect on sperm function

The ability of sucrose to protect spermatozoa against mitochondrial damage, artificial acrosome reaction and DNA fragmentation during ultra‐rapid cryopreservation in canine sperm was investigated. Swim‐up selected spermatozoa of second‐fraction semen were vitrified with different concentrations of sucrose (0.1, 0.25 and 0.4 m ) in proportion 1 : 1 v/v with HTF–BSA 1%. From each group, 30‐μl suspensions of cells were dropped directly into liquid nitrogen and stored for at least 24 h. Cells were thawed by submerging the spheres in HTF with 1% BSA at 37 °C. The number of progressively motile spermatozoa was significantly higher in the sucrose 0.25 m + HTF–BSA 1% (42.5 ± 2.3%, P < 0.01) than in HTF only (1.66 ± 0.3%). The same combination of sucrose 0.25 m + HTF–BSA 1% (42.7 ± 1.5%) had a stronger cryoprotective effect on the integrity of mitochondrial membrane potential (P < 0.05) and decreased the DNA fragmentation (2.8 ± 0.5%) as compared with HTF only (1.93 ± 0.6% and 5.6 ± 0.6% respectively). With respect to acrosome‐reacted spermatozoa, no significant difference was found between the groups investigated (P > 0.05). It is concluded that sucrose, a nonpermeable cryoprotectant, can effectively preserve important physiological parameters such as mitochondrial membrane potential and DNA integrity during ultra‐rapid cryopreservation.     

Male Subfertility and the Outcome of Intrauterine Insemination/Der Einfluß männlicher Fertilitätsstörungen auf die Effektivität der intrauterinen Insemination

Seven out of 27 infertile women conceived by intrauterine insemination (IUI, 26%), one of them twice. Three other pregnancies occurred spontaneously after the discontinuation of treatment. A comparison of the data obtained from 65 treatment cycles revealed that the number of motile spermatozoa per ml of ejaculate and per ml of medium after swim-up preparation was higher in the ultimately fertile group as compared to the patients who failed to conceive. It is concluded that male subfertility affects the outcome of IUI unfavorably.     

Lycopene and resveratrol improve post‐thaw bull sperm parameters: sperm motility,mitochondrial activity and DNA integrity

We focussed on evaluating the protective effect of lycopene and resveratrol on post‐thaw bull sperm and oxidative stress parameters. Nine ejaculates for each bull were used in the study. Each ejaculate, splitted into three equal aliquots and diluted at 37 °C with base extenders containing lycopene (1 × 10^?3 g ml^?1) and resveratrol (1 mm ), and no antioxidant (control), was cooled to 5 °C and then frozen. Frozen straws were thawed in a water bath for evaluation. The supplementation of the semen extender with lycopene and resveratrol increased the percentages of post‐thawed computer‐assisted sperm analysis (CASA) motility (55.8 ± 3.8 and 61.9 ± 4.0%) and progressive motility (38 ± 2.4 and 37 ± 8.8), compared with the controls (50.7 ± 2.65 and 33.3 ± 3.74%, respectively, P < 0.05). Resveratrol provided a higher ALH (4.3 ± 0.1), in comparison with the control (3.9 ± 0.3, P < 0.05). The supplementation of the semen extender with lycopene and resveratrol produced a higher mitochondrial activity (24.6 ± 2.9 and 30.1 ± 6.5% respectively), compared with that of the control (11.8 ± 9.5%, P < 0.05). It was determined that both antioxidants resulted in a lower percentage of sperm with damaged DNA than that of the control (P < 0.05). Sperm motion characteristics except for ALH, acrosome integrity, sperm viability and oxidative stress parameters were not affected by the adding of lycopene and resveratrol.     

Effects of caffeine supplementation in post‐thaw human semen over different incubation periods

This study aimed to evaluate the effects of caffeine supplementation in post‐cryopreservation human semen over different incubation periods. After collection by masturbation, 17 semen samples were analysed according to World Health Organization criteria, processed and cryopreserved with TEST‐yolk buffer (1 : 1) in liquid nitrogen. After a thawing protocol, samples were incubated with 2 mm of caffeine for 0, 5, 15, 30 or 60 min, followed by analysis of motility and mitochondrial activity using 3,3′‐diaminobenzidine (DAB). Mean variance analysis was performed, and P < 0.05 was the adopted significance threshold. Samples incubated for 15 min showed increased progressive motility compared to other periods of incubation, as well as a reduced percentage of immotile spermatozoa (P < 0.05). In samples incubated for 5 min, increased mitochondrial activity above 50% was observed (DABI and DABII). Although cryosurvival rates were low after the cryopreservation process, incubation with caffeine was associated with an increase in sperm motility, particularly 15‐min incubation, suggesting that incubation with caffeine can be an important tool in patients with worsening seminal quality undergoing infertility treatment.     

弗司扣林对人精子运动功能的影响

目的 通过研究弗司扣林(forskolin)对体外人精子的运动功能有无影响，了解环磷酸腺苷／蛋白激酶A(cAMP／PKA)信号传导途径是否参与人精子运动功能的调节。方法 10例健康成年男性手淫获得新鲜精液，经上游优化处理后与不同浓度的forskolin一起孵育20、30、60min后，采用计算机辅助的精子分析系统(CASA)检测精子的各项运动参数，并进行对比分析。结果 forskolin在体外能显著提高人精于的活率及前向性运动百分率，而对精子的形态及直线速度(VSL)和曲线速度(VCL)无明显影响。结论 forskolin在体外能提高人精子的运动功能，此结果为证实cAMP/PKA信号传导通道参与调节人精子运动功能提供了一定的实验依据。     

The influence of relaxin on motility of human sperm in vitro

Sperm of healthy men were incubated in an IVF medium with relaxin at concentrations of 3, 30, 300 and 3000 ng ml-1. Immediately after addition of relaxin and 60 and 120 min later motility, progressive motility, mean path velocity, mean progressive velocity, mean linearity and mean lateral head displacement were measured with the Hamilton-Thorn motility analyser. Neither immediately after relaxin addition, nor after 60 or 120 min, was an improvement of sperm motility observed at any concentration.     

Transforming growth factor βreceptor Ⅱ expression in experimental cryptorchidism and apoptosis in spermatogenic cells in rats

Objective: To evaluate the sensitivity of sperm motility assay for detecting the endotoxin effect on human sperm in vitro. Methods: Motile human sperm were separately incubated for up to 24 hours with different concentrations of endotoxin (0.5, 1, 10, 1000, 10 000 and 50 000 ng/mL). Then the sperm motility was determined. The effect of endotoxin on the sperm motility in media without albumin was also determined. In addition, at the endotoxin concentrations of 0.5, 1 and 10 ng/mL, the sensitivity of the assay was compared to those of 1-cell and 2-cell mouse embryo bioassays. Results: At levels of 0.5-1 000 ng/mL endotoxin in media with 2 mg/mL albumin, sperm did not show significant change in motility after 24 h of incubation (P>0.05), while it was significantly inhibited at endotoxin levels of 10 000 and 50 000 ng/mL. In media without albumin, endotoxin levels of 50 000 and 1 000 ng/mL, markedly inhibited the sperm motility after 2 or 8 h of incubation (P<0.01). With media containing 0.5 and 1 ng/mL endo     

不采用冷冻保护剂的玻璃化法与常规冷冻法对人精子冷冻复苏效果的比较

目的比较不采用冷冻保护剂的玻璃化法与常规冷冻法对人精子冷冻复苏的效果。方法将15份上游后的精液标本分别采用常规精子冷冻和不使用冷冻保护剂的冷冻环玻璃化法冷冻,比较精子复苏后的活力、形态及精子膜的完整性三项指标。结果冷冻前、前向活动精子百分率、正常精子形态百分率及精子膜完整率分别为(79±6.42)%、(34±9.36)%和(91±5.18)%;不采用冷冻保护剂的玻璃化法冷冻复苏后,三者分别为(42.20±8.35)%、(31.00±7.63)%和(50.00±9.34)%。常规冷冻法冷冻复苏后,三者分别为(38.00±15.80)%、(30.00±5.24)%和(47.00±13.67)%。冷冻前后前向活动精子百分率和精子膜完整率的差别有统计学意义,但两种冷冻方法相比差异无统计学意义。结论使用不加入冷冻保护剂的玻璃化方法冷冻人的精子是可行的,可取得与常规冷冻相同的效果。     

睾酮体外对人精子运动参数的影响

目的通过研究睾酮体外对人精子运动参数的影响,探讨睾酮在男性不育症治疗中的作用。方法10例健康生育男性手淫获得精液,经上游优化处理后的精子与不同浓度的睾酮孵育10、30、60min,10例弱精子症患者手淫取精并与睾酮孵育10、60、120min后,采用计算机辅助的精液分析系统(CASA)检测精子的运动参数。结果50ng/dl(1.73nmol/L)睾酮在体外能显著增强正常人精子的直线速度(VSL)、曲线速度(VCL)和平均速度(VAP),而对活率、前向运动百分率无明显影响,而100ng/dl(3.47nmol/L)睾酮使精子活率和前向性运动百分率均有明显下降(P<0.01);1.04nmol/L～1.73nmol/L浓度睾酮能显著增强弱精子症患者精子的活率、前向运动百分率及VSL,并随着浓度的增加,睾酮作用显著增强,而对VCL和VAP无明显影响。结论低浓度(1.04nmol/L～1.73nmol/L)睾酮在体外显著增高弱精症患者精子的运动参数。     

rhGM-CSF体外对人精子运动参数的影响

目的观察体外添加不同浓度的重组人粒细胞-巨噬细胞集落刺激因子(rhGM-CSF)对人精子运动参数的影响,探讨其在精子运动中的作用机制。方法健康生育男性和弱精子症患者各10例手淫取精,经简易上游优化处理后的精子与不同浓度rhGM-CSF溶液孵育10min、30min、60min后,采用计算机辅助的精液分析系统检测精子各项运动参数的变化。结果对精液运动参数正常的标本,1ng/ml和10ng/ml的rhGM-CSF溶液能显著提高精子的活率、前向性运动百分率,在10ng/ml时平均直线运动速度也有显著提高。对弱精子症患者精液标本,1ng/ml和10ng/ml的rhGM-CSF溶液能显著提高精子的前向运动百分率和直线运动速度,10ng/ml的rhGM-CSF对精子活率和平均路径速度也有显著性提高。两类精子的曲线运动速度均无显著性变化。结论1ng/ml～10ng/ml浓度范围的rhGM-CSF体外能显著改善精子的运动功能。     

Stimulation of sperm motility by kinins in fresh and 24 hours aged human ejaculates

Kallikrein, a kinin-releasing proteinase, significantly stimulates human sperm motility in fresh ejaculates with primarely reduced sperm motility (asthenozoospermia) as well as in 24 hours stored ejaculates with reduced sperm motility resulting from post-ejaculatory aging processes. adiition of a kininogen source (serum) further significantly improved sperm motility of kallikrein-stimulated ejaculates. In contrast to the 24 hours lasting stimulation of kallikrein, enhancement of sperm motility by kinins is limited to 2 hours after addition of a single dose of kallidin (1 ng/ml).     

己酮可可碱对精子活力的体外改善作用

目的体外分析己酮可可碱(Pentoxifylline，PF)对精子活力的改善作用，探讨PF最佳作用浓度和时间，为实施人工授精奠定基础。方法本实验就PF的不同浓度(0．6mmol／L、3．0mmol／L)和不同时间(30min，60min)，恒温下与70例弱精子的体外孵育，并与对照比较，观察精子活力特性的改善情况。结果PF的不同浓度及不同时间对精子活力均有改善作用，而用PF的0．6mmol／L浓度、孵育30min，对体外弱精子的活力有显著增强作用。结论PF对体外弱精子的活力有改善作用，药物浓度和作用时间直接影响孵育效果。     

Validation of a single-step procedure for the objective assessment of sperm motility characteristics

A relatively cheap method is described for the objective assessment of sperm concentration and motility characteristics. The method uses a digitizing tablet with cursor, a micro-computer and a phase-contrast or dark-field microscope equipped with a drawing tube. With this technique the following are accurately assessed: sperm concentration, percentage motility, motility grading, concentration of grade a motile spermatozoa, sperm velocity, linear velocity, linearity and angularity. The data are acquired in less than 5 min. Validation studies reveal this method to be accurate, reproducible (coefficient of variation of motility characteristics = less than 7%) and clinically useful.     

光量子体外照射对精子活力的影响

目的 观察光能对精子活力的影响,探索一条体外不去精浆、提高精子活力的方法。方法 通过对54例因精子活力低下引起男性不育患者的精液,体外应用SB-99型血液处理器进行UV(紫外线)照射,观察同份精液在处理前后活动精子百分率和应用激光光散射测定精子前向运动速率,同时作细菌培养和细菌计数,并作精浆SOD和精浆粘稠度测定。结果 精子速率、活动精子百分率、活力得分、精浆粘稠度及SOD活性处理后较处理前均有明显改善(P<0.01),当UV>500J/cm~3以上,UV照射不同剂量组间精子活力无显著差异(P>0.05);处理最适宜的时间为:3min1000J/cm~3在3～5min时已显示杀菌作用。结论 UV照射引起精浆粘稠度降低、杀灭细菌和SOD活性增加,可能是其提高精子活力的原因。     

Effect of zinc on human sperm motility and the acrosome reaction

This study has assessed the effect of zinc on human sperm motility and the acrosome reaction in vitro. Progressively motile human sperm were selected by swim-up and by glass bead columns and then incubated in a medium in which capacitation happened in an asynchronous way. Different doses of zinc (1, 10, 100 and 1000 microM) were added for periods of 2, 4 or 6 h. Other samples were incubated with zinc (1000 microM), and after 1 h incubation, the zinc was removed. Aliquots of each culture were used to evaluate progressive motility and the acrosome reaction using a triple-stain technique. Sperm motility was reduced when the amount of zinc added was greater than or equal to 100 microM, and these doses also caused a significant reduction in the % of sperm undergoing the acrosome reaction. After removal of zinc and further incubation in zinc-free medium for 1 h, an increase in the percentage of motile and acrosome-reacted sperm was observed. However, the increase in acrosome reaction did not reach the values observed in controls. Results suggest that extracellular zinc acts as an inhibitor of human sperm motility and the acrosome reaction (and/or capacitation and the acrosome reaction). This inhibitory effect is reversible and occurs in a dose-dependent fashion. The probable mechanisms involved are discussed.     

Caffeine- and kallikrein-induced stimulation of human sperm motility: a comparative study

Stimulation of human sperm motility by caffeine and kallikrein was compared in the same semen material. Caffeine, a cyclic nucleotide phosphodiesterase inhibitor, induced an immediate stimulation of sperm motility the intensity being relatively constant during the first two hours of incubation. Kallikrein, a kinin-releasing proteinase, induced a similar enhancement of total sperm motility, but showed a delayed type of reaction with maximum stimulation at 2 hours of incubation. In contrast to the effect of caffeine lasting some hours, enhancement of sperm motility induced by kallikrein was observed 24 hours. Simultaneous addition of serum (kininogen source) and kallikrein to semen samples led to a sitmulation of total sperm motility with higher mean values than those obtained by caffeine of kallikrein alone. However, the ratio of spermatozoa with very good forward progression was highest during caffeine stimulation. Simultaneous addition of caffeine and kallikrein led to a further improvement of sperm motility which was significantly above that produced by caffeine or kallikrein alone. This observation and the finding of a different response of the spermatozoa of two ejaculates towards caffeine of kallikrein indicate that caffeine (cyclic AMP) interferes quite differently in comparison to kallikrein (kinins) in stimulating sperm motility.     

Hypotonic challenge reduces human sperm motility through coiling and folding of the tail

Human ejaculates collected for in vitro procedures show variably rapid increases in osmolality, depending on enzymatic degradation of compounds. Changes in osmolality can affect cell functions due to the energy consuming processes needed to control cell volume. The aim was to examine the effects of a hypotonic challenge for spermatozoa exposed to increased osmolality. Spermatozoa were selected by density gradient centrifugation and washed in media with different osmolalities. Osmolality was measured by freezing-point depression and sperm velocities by CASA. Swimming pattern observations and assessments of tail morphology of fixed spermatozoa were done with phase contrast microscopy. Increased osmolality did not change the curvilinear velocity (VCL), while decreased osmolality reduced or abolished VCL nonreversibly. For spermatozoa first exposed to 400 mOsm/kg, reversal of osmolality to 290 mOsm/kg reduced the VCL and the average path velocity (VAP) permanently. Hypotonic challenges increased sperm tail coiling and folding in a dose-response pattern. Spermatozoa once adjusted to high osmolality in the liquefied ejaculate are likely to suffer if exposed to a medium with a lower osmolality. For improved success of Assisted Reproductive Technologies (ART), it appears to be important to minimise the duration of sperm exposure to the ejaculate, by early dilution or sperm preparation.     

The influence of sperm density on the motility characteristics of washed human spermatozoa

To study the effects of sperm density on the results of computer-assisted semen analysis (CASA), 10 washed semen samples were diluted and measured with the CellTrak/S CASA system in a concentration range of 10–180×10⁶ spermatozoa/ ml. All sperm motility parameters were influenced to some extent by sperm density. The motility percentage was influenced significantly in 5 samples ( P< 0.005), the straight line velocity in all samples (P<0.0005 in 7 samples), the curvilinear velocity in 3 samples ( P< 0.005), the linearity in 9 samples (P<0.0005 in 6 samples) and the lateral head displacement in 9 samples (P< 0.005 in 6 samples). In general, the CellTrak/S data are influenced significantly if sperm density exceeds 50 × 10⁶ spermatozoa/ml.
The influence of sperm density on the motility parameters can be explained both by the accuracy of the CASA system and by actual changes in the motility of the spermatozoa. In the light of other published studies, it is concluded that sperm motility measurements with CASA systems should be assessed using 25–50 × 10⁶ spermatozoa/ml, especially in studies concerning lateral head displacement and the linearity, as in sperm hyperactivation studies.     

阿片类镇痛药体外对精子运动的影响

目的:观察阿片类镇痛药体外对人精子运动功能的影响。方法:试验选取20例精子活力正常的精液标本,每例精液均一式19份,1份为对照,余分别与3种浓度下的6种不同阿片类镇痛药于37℃温箱孵育4 h,分别于15 min(t1)、2 h(t2)、4 h(t3)3个时间点用计算机辅助精液分析系统(CASA)分析精子活力。结果:①1×10-5、2×10-3、0.05 mg/ml芬太尼作用的精子,其3个时间点的(a+b)级精子百分率与对照组相比均显著降低(P<0.05);②1×10-5、2×10-3、0.05 mg/ml阿芬太尼作用的精子,其3个时间点的(a+b)级精子百分率与对照组相比均显著降低(P<0.05);③1×10-5、2×10-3、0.05 mg/ml舒芬太尼作用的精子,其3个时间点的(a+b)级精子百分率与对照组相比均显著降低(P<0.05);④1×10-5 mg/ml布托啡诺作用的精子,其3个时间点的(a+b)级精子百分率与对照组相比均无显著变化(P>0.05),2×10-3、0.05 mg/ml布托啡诺3个时间点的(a+b)级精子百分率与对照组相比均显著降低(P<0.05);⑤1×10-5 mg/ml地佐辛作用的精子,其3个时间点的(a+b)级精子百分率与对照组相比均无显著变化(P>0.05),0.05、0.5 mg/ml地佐辛3个时间点的(a+b)级精子百分率与对照组相比均显著降低(P<0.05);⑥3×10-5、0.05 mg/ml喷他佐辛处理精子,其3个时间点的(a+b)级精子百分率与对照组相比均无显著变化(P>0.05),而0.5 mg/ml喷他佐辛3个时间点的(a+b)级精子百分率与对照组相比均显著增加(P<0.05);⑦0.05 mg/ml布托啡诺作用精子15 min可产生精子活力完全抑制(制动效应),以及2×10-3mg/ml布托啡诺作用精子2 h,0.05、0.5 mg/ml地佐辛作用精子2 h时均产生制动效应;而芬太尼、阿芬太尼、舒芬太尼在0.05 mg/ml作用精子并未发现此效应。⑧0.05 mg/ml舒芬太尼、布托啡诺、地佐辛、芬太尼、阿芬太尼作用精子15 min活力衰减程度:舒芬太尼、布托啡诺、地佐辛与阿芬太尼相比均有显著性的差异(P<0.05),芬太尼与阿芬太尼相比无显著性差别(P>0.05)。结论:不同阿片受体镇痛药在相同作用时间内,低浓度布托啡诺、地佐辛对精子运动无影响,高浓度呈现完全抑制精子活力,而相同低浓度芬太尼、阿芬太尼、舒芬太尼均显著抑制精子活力,所测相同浓度范围内仅部分抑制精子活力。与此相反,高浓度喷他佐辛可促进精子运动。     

The routine assessment of sperm motility at room temperature and 37°C

The World Health Organization (WHO) laboratory manual (1992) states that assessment of sperm motility can be performed at either 37^OC or room temperature (20–24^OC). The motility of spermatozoa in 44 semen samples (22 fresh samples and 22 frozen-thawed samples) was assessed at both of these temperatures and a significant difference in the motility profiles was noted, specifically an increase at 37^OC in the percentage (expressed here as median and ranges) of spermatozoa with excellent progressive motility and an overall increase in the percentage with total progressive motility. With fresh samples the excellent progressive motility increased from 41 (19–53) to 54 (30–66) and the overall motility from 58.5 (39–74) to 65.0 (40–79). With the frozen—thawed samples the excellent motility increased from 14 (1–33) to 25 (6–45) and the overall motility from 30.5 (14–51) to 33.0 (16–52). As the WHO laboratory manual was published. 'In response to a growing need for the standardisation of procedures for the examination of human spermatozoa' it is proposed that only one temperature for routine analysis should be used, namely 37^OC, which may have more physiological relevance and eliminate effects of fluctuations in ambient laboratory temperature.